Abstract
BackgroundMost of the proteases classified into the M23 family in the MEROPS database exhibit staphylolytic activity and have potential as antibacterial agents. The M23 family is further classified into two subfamilies, M23A and M23B. Proteases of the M23A subfamily are thought to lack the capacity for self-maturation by auto-processing of a propeptide, which has been a challenge in heterologous production and application research. In this study, we investigated the heterologous expression, in Bacillus subtilis, of the Lysobacter enzymogenes beta-lytic protease (BLP), a member of the M23A subfamily.ResultsWe found that B. subtilis can produce BLP in its active form. Two points were shown to be important for the production of BLP in B. subtilis. The first was that the extracellular proteases produced by the B. subtilis host are essential for BLP maturation. When the host strain was deficient in nine extracellular proteases, pro-BLP accumulated in the supernatant. This observation suggested that BLP lacks the capacity for self-maturation and that some protease from B. subtilis contributes to the cleavage of the propeptide of BLP. The second point was that the thiol-disulfide oxidoreductases BdbDC of the B. subtilis host are required for efficient secretory production of BLP. We infer that intramolecular disulfide bonds play an important role in the formation of the correct BLP conformation during secretion. We also achieved efficient protein engineering of BLP by utilizing the secretory expression system in B. subtilis. Saturation mutagenesis of Gln116 resulted in a Q116H mutant with enhanced staphylolytic activity. The minimum bactericidal concentration (MBC) of the wild-type BLP and the Q116H mutant against Staphylococcus aureus NCTC8325 was 0.75 μg/mL and 0.375 μg/mL, respectively, and the MBC against Staphylococcus aureus ATCC43300 was 6 μg/mL and 3 μg/mL, respectively.ConclusionsIn this study, we succeeded in the secretory production of BLP in B. subtilis. To our knowledge, this work is the first report of the successful heterologous production of BLP in its active form, which opens up the possibility of industrial use of BLP. In addition, this study proposes a new strategy of using the extracellular proteases of B. subtilis for the maturation of heterologous proteins.
Highlights
Most of the proteases classified into the M23 family in the MEROPS database exhibit staphylolytic activity and have potential as antibacterial agents
Secretory expression of beta-lytic protease (BLP) by B. subtilis We investigated the possibility of heterologous secretory expression of BLP (GenBank: BAV99603.1) derived from L. enzymogenes M497-1 [25] using B. subtilis strain 168 as a host
Three types of expression plasmids were used: one encoding a full length BLP, including the signal, pro, and mature sequence; a second encoding the pro and mature sequence of BLP fused to the signal sequence of Egl-237; and a third encoding the mature sequence of BLP fused to the signal sequence of Egl-237 (Fig. 1)
Summary
Most of the proteases classified into the M23 family in the MEROPS database exhibit staphylolytic activity and have potential as antibacterial agents. Many of the enzymes in this family have glycylglycine endopeptidase activity, which cleaves peptide linkers that cross-link cell wall peptidoglycans to lyse Gram-positive bacteria such as staphylococci [2]. Since these enzymes have the activity to kill pathogenic Staphylococcus aureus, which has a pentaglycine linker in its cell wall peptidoglycan, these proteins are expected to find application as antimicrobial agents in medicine, veterinary science, and the food industry [3]. Among the M23 family members, lysostaphin in particular has been extensively studied for application as an antimicrobial agent; this enzyme has been shown to be effective in a number of preclinical animal models and to show efficacy in a small number of clinical trials [10]. Because LasA protease has broader substrate specificity than lysostaphin [12, 13], it may be effective in the treatment of opportunistic infections caused by staphylococci other than S. aureus [3]
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